Biology subjects

Wein, M. N.

Publications and source records attributed to Wein, M. N..

8 recordsLinked to original sources

bioRxiv2026

CRTC, not phosphorylated CREB1, drives cAMP-induced transcription across diverse cell types

Specialized cell types layer cell-type-restricted proteins, metabolites, and organelles onto a shared foundation of core cellular processes. How this ubiquitous machinery generates lineage-specific outputs is central to both cell biology and therapeutic development. G-protein-coupled receptors (GPCRs) respond to receptor-restricted ligands to drive cell-type-specific transcription through cAMP-mediated activation of protein kinase A (PKA), which activates the transcription factor CREB1 through two parallel modes: direct phosphorylation at serine 133, and inhibition of salt-inducible kinases (SIK) that restrain the CRTC coactivators. How these modes integrate and contribute to signaling across cell types has remained unresolved, obscured by genetic redundancy and essentiality. Here we combine focused genetic analyses with a cross-lineage transcriptomic survey to dissect these parallel inputs. In Creb1/Atf1/Crem triple-knockout cells, a non-phosphorylatable mutant CREB1S133A fully rescued endogenous target gene activation, while Crtc1/Crtc2/Crtc3 ablation abolished transcription even with intact CREB1 serine 133 phosphorylation. As part of this mechanism, we found the annotated repressor ICER can instead act as a positive regulator, substituting for full-length CREB1 paralogs to drive a feedforward loop. Across melanocytes, hepatocytes, osteocytes, macrophages, and neurons, SIK inhibition recapitulated cAMP-PKA-driven transcription across both shared and cell-type-specific gene expression programs, with neurons a notable exception. These results invert the canonical model, placing CRTC recruitment as the dominant driver of CREB1-mediated transcription across diverse lineages, reframing how cAMP-PKA signaling can be interpreted and therapeutically targeted.

cell biology↗

bioRxiv2026

Intermittent parathyroid hormone employs autonomous and non-autonomous mechanisms to drive osteogenesis from Ebf3-expressing skeletal progenitor cells

How systemic hormonal signals coordinate stem cell fate decisions in adult tissues remains incompletely understood. In bone marrow, Cxcl12-abundant reticular (CAR) cells, marked by Early B-cell Factor 3 (Ebf3) expression, are multipotent mesenchymal progenitors that maintain the hematopoietic stem cell niche and serves as a major osteoblast progenitor source during adult bone remodeling. Using inducible lineage tracing coupled with single-cell transcriptomics and conditional genetics in mice, we show that intermittent parathyroid hormone (iPTH; teriparatide) drives osteogenesis from CAR cells by simultaneously engaging cell-intrinsic and cell-extrinsic mechanisms. Directly, iPTH suppresses lineage-enforcing transcription factors Ebf3, Ebf1, and Foxc1, thereby destabilizing progenitor identity and priming CAR cells for osteogenic commitment. Simultaneously, iPTH stimulates osteoclastic bone resorption, releasing TGF{beta} which recruits these primed progenitors to bone surfaces, a process abolished by osteoclast depletion. Preventing CAR cell maturation via Sp7 deletion abrogates iPTH-induced bone gain, establishing these progenitors as essential mediators of bone anabolism. This coupled mechanism, in which intrinsic transcriptional priming converges with extrinsic niche remodeling, is conserved in human CAR cells from teriparatide-treated postmenopausal women, which show concordant suppression of EBF3 and FOXC1 and elevated TGF{beta}-responsive gene signatures. These findings reveal a general principle by which a systemic hormone orchestrates tissue remodeling through simultaneous reprogramming of progenitor identity and remodeling of the niche microenvironment.

physiology↗

bioRxiv2025

Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

Osteocytes play critical roles in bone, making them attractive targets for therapeutics to improve bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin {beta}3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify a role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.

cell biology↗

bioRxiv2024

Trafficking and translation of mRNA in osteocyte dendrites

Osteocyte dendrites extend great distances through canaliculi in bone supported by an extensive cytoskeletal network. The formation and maintenance of osteocyte dendrites is closely linked to bone health, yet the mechanisms regulating these processes are poorly understood. Here we tested the hypothesis that osteocytes transport mRNA to their dendrites for local translation as a mechanism of regulating the formation and maintenance of these subcellular structures. Using molecular and imaging approaches, we demonstrated that a subset of mRNAs are enriched in Ocy454 osteocyte-like cell dendrites. To understand how certain mRNAs are directed to dendrites, we performed a massively parallel reporter assay with 7115 oligonucleotide sequences designed from the 3 untranslated regions of eight dendrite-enriched transcripts. We identified localization sequences within some 3UTRs that were both sufficient and necessary for trafficking to osteocyte dendrites, indicating that localization directions are specifically encoded in enriched transcripts. Furthermore, we found that dendrites contain the elements necessary for local translation, and trafficked transcripts were engaged in active translation within dendrites. Together this work describes a molecular regulatory mechanism novel to osteocyte biology with many potential consequences for the spatiotemporal control of the cytoskeleton and dendrites.

molecular biology↗

bioRxiv2024

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gs, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gs and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

molecular biology↗

bioRxiv2021

Partial prevention of glucocorticoid-induced osteocyte deterioration with osteocrin gene therapy

Glucocorticoid (GC)-induced osteoporosis and subsequent bone fragility are preceded by death and dysfunction at the cellular level. In particular, short-term glucocorticoid excess suppresses osteocyte remodeling of the surrounding bone mineral, causes apoptosis of osteoblasts and osteocytes, and disrupts homeostatic bone remodeling. Preventing apoptosis and preserving osteocyte morphology and function could be effective means of preventing bone loss during glucocorticoid excess. We hypothesized that osteocrin, which preserves osteocyte viability and morphology in other models where osteocyte defects exist, could prevent osteocyte death and dysfunction in a GC excess model. We used a liver-targeted adeno-associated virus (AAV8) to induce osteocrin overexpression in mice one week prior to implantation with prednisolone or placebo pellets. After 28 days, tissues were collected for micro-CT and histological analysis. GC excess caused the expected reduction in cortical bone thickness and osteocyte canalicular length in control AAV8-treated mice, and these effects were blunted in mice overexpressing osteocrin. However, GC-induced changes in cortical porosity, trabecular bone mass, and gene expression were not prevented by osteocrin. While the mechanism of osteocrins effects on osteocyte morphology warrants further investigation, this study does not support a role for this model of osteocrin supplementation to combat the full skeletal effects of GC excess.

physiology↗

bioRxiv2021

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin

Osteocytes use an elaborate network of dendritic connections to control bone remodeling. Some osteoblasts embed within mineralized bone matrix, change shape, and become osteocytes. The molecular circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7, a gene linked to rare and common skeletal disease, in mature osteoblasts and osteocytes causes severe defects in osteocyte dendrites. Unbiased profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues phenotypic and molecular defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates overt defects in osteocyte maturation in vivo in the absence of Sp7. Sp7-dependent gene networks enriched in developing osteocytes are associated with rare and common human skeletal traits. Moreover, humans homozygous for the osteogenesis imperfecta-causing SP7R316C mutation show dramatic defects in osteocyte morphology. Genes that mark osteocytes in vivo and that are regulated by Sp7 in vitro are highly enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. Taken together, these findings reveal a crucial role for Sp7 and its target gene Osteocrin in osteocytogenesis, demonstrating that pathways that control osteocyte development influence human bone diseases.

molecular biology↗

bioRxiv2021

Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption

Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.

physiology↗